HPV infection is highly prevalent among young, sexually active male and female individuals. Large prospective studies showed that acquisition of HPV from male partners is common, occurring in 40-60% of subjects during a 3 year follow-up period (Koutsky et al., 1997, Ho et al., 1998, Marrazzo et al., 2000). Therefore, HPV is probably the most common sexually transmitted disease.
Papillomaviruses of the high-risk types (e.g. HPV16, 18, 31, 33, and 45) are responsible for cervical cancer (Bosch et al., 1995, Zur Hausen, 1996). Following infection of the basal epithelial cells, the immediate HPV early genes E1, E2, E5, E6 and E7 are expressed. The E1 and E2 genes regulate viral replication. Furthermore, the E2 protein controls the expression of the E6 and E7 oncoproteins. The E6 protein of the high-risk HPV types specifically binds to p53 and targets its rapid degradation through the ubiquitin pathway. P53 is involved in initiation of apoptosis and loss of this protein result in the prevention of apoptosis (Scheffner et al., 1990). The E7 protein of high-risk types binds to pRB, which normally prevents cells from entering the cell cycle by inactivating E2F, a protein needed for cell cycle entry (Dyson et al. 1989). E7 expression results in the failure of infected cells to withdraw from the cell cycle and differentiate.
Prolonged and elevated expression of the E6 and E7 oncoproteins is tightly associated with HPV-induced dysplasia and transformation into cervical carcinoma.
The protective role of the immune system in the defense against HPV-related diseases and HPV-induced cancer in humans is suggested by the fact that compared to normal controls, immunosuppressed renal transplant patients and patients infected with HIV display a 17-fold greater incidence of genital HPV infection (Ho et al., 1994, Matorras et al. 1991, Halpert et al. 1986). The diminished capacity of immunosuppressed individuals to resolve HPV infection indirectly points towards a protective role of the immune system early in infection. Evidence of protection against HPV via immunity against the early antigens E2, E6 and E7 comes from the cottontail rabbit papillomavirus model which is the major animal model for cancer-associated papillomaviruses. Vaccination with the nonstructural proteins E1 and E2 induces the regression of virus-induced papillomas whereas viral tumor growth is suppressed.
Furthermore, rabbits vaccinated with the combination of the E1, E2, E6, and E7 genes were completely protected against viral challenge (Han et al. 1999, Selvakumar et al. 1995). Importantly, rabbits with progressively growing papilloma virus-induced warts could eradicate their lesions as well as clear latent viral infections following two injections with a vaccine comprising E6 and E7 long overlapping peptides (Vambutas, Vaccine 2005). These data indicate that immunity against E2, E6, and E7 can be effective as immunoprophylaxis of papillomavirus infection as well as therapeutically for HPV induced lesions and cancer.
Considerable interest exists in the identification of epitopes involved in the immune response to HPV16, given the possibility to incorporate these as subunits into a vaccine or to use these epitopes to monitor vaccine induced immunity in vivo. Since most epithelial cells express MHC class I but not class II, the attention has so far been focused on the induction of tumoricidal HPV-specific CD8+ cytotoxic T lymphocytes (Melief et al., 2000; Ressing et al., 1995; Ressing et al., 2000; Ressing et al., 1996). HPV specific CD8+ T-cell reactivity has been found in the peripheral blood of patients diagnosed with cervical intraepithelial neoplasia grade III (CIN III) lesions or cervical carcinoma (Nimako et al., 1997; Ressing et al., 1996) and in tumor-infiltrating T-cell populations isolated from patients with cervical cancer (Evans et al., 1997). Tumor specific CD4+ T helper (“Th”) immunity is now also considered pivotal for the efficient eradication of solid tumors, despite the fact that most of these tumors do not express MHC class II (reviewed in Melief et al., 2000; Pardoll and Topalian, 1998; Toes et al., 1999). Recent evidence indicates that CD4+ tumor specific T-cells are required not only for optimal induction of CD8+ tumor specific CTL but also for optimal exertion of local effector cell function by these CTL (Ossendorp et al., 1998, Toes et al., 1999). For induction of MHC class I restricted tumor-specific immunity, cross-presentation of antigens that have been captured by professional antigen presenting cells appears to play a dominant role. For proper induction of an effective tumor-specific CTL by cross-priming tumor-specific CD4+ T cell help is required (Toes et al., 1999, Schoenberger et al., 1998).
Strong indications for a protective role of HPV-specific Th-immunity was suggested by the predomination of CD4+ T-cells in regressing genital warts (Coleman et al., 1994) as well as by the detection of delayed-type hypersensitivity responses to HPV16 E7 in the majority of subjects with spontaneous regressing CIN lesions (Hopfl et al., 2000). Furthermore, in most healthy persons the immune system succeeds in eliminating the virus before malignancies develop (Koutsky, 1997; Evander, 1995). In line with this, more that half of all healthy females tested display strong proliferative HPV16 E2- and E6-specific Th1/Th2 cell memory responses (de Jong, 2002; Welters, 2003; de Jong, 2004). Furthermore, Th-reactivity against E2 was found to occur at time of viral clearance (Bontkes, 1999). Healthy subjects display HPV16 E7-specific immunity too (Welters, 2003; van der Burg, 2001). In contrast, the occurrence of HPV-induced cancer is strongly associated with immune failure. Analysis of HPV16 E2-, E6- and E7-specific CD4+ T-cell immunity in the peripheral blood of patients with HPV16+ induced neoplasia revealed that half of the patients with high grade vulvar neoplasia (van Poelgeest, 2005) and the majority of patients with CIN III failed to mount a proper immune response (de Jong, 2004). Of the cervical carcinoma patients tested, approximately half lacked any detectable proliferative T-cell responses. The other half displayed weak proliferative HPV16 E2- and E6-specific T-cell responses not associated with the production of Th1/Th2 cytokines but with IL-10 (de Jong, 2004). This corroborates previous observations that E6- and E7-specific proliferative responses can be present (Luxton, 2003), but that the peripheral Th1 response in cervical carcinoma patients is low (de Gruijl, 1996; de Gruijl, 1998) or lacking (Tsukui, 1996). Because, the CD4+ T-cell response is of pivotal importance for the induction and maintenance of CD8+ CTL immunity (Melief, 2002), these data offer a plausible explanation why peripheral HPV16-specific CTL are rarely detected in patients with high-grade dysplasia or cancer (Ressing, 1996; Bontkes, 2000; Nimako, 1997; Youde, 2000), while such CTL are more commonly detectable in women without HPV16+ neoplasia (Nakagawa, 1997; Nakagawa, 1999).
For a clinically relevant approach of immunizing subjects against HPV in particular, it is preferred that both specific T-helper cells and CTL are induced. We have already shown that immunization with minimal CTL epitopes results in protection against tumors in some models (Kast et al. 1991) whereas, in other models, it can lead to tolerance or functional deletion of virus- and tumor-specific CTL that when otherwise induced are protective (Toes et al. 1996ab). The occurrence of tolerance or functional deletion decreases the effects of vaccination significantly. Epitopes involved with this effect were therefore not suitable for immunization purposes. Processing of exogenous antigens for presentation by MHC class 1 molecules by cross-priming as well as by other mechanisms is now widely recognized second pathway of processing for presentation by MHC class T, next to the well known endogenous route (Jondalet al. 1996, Reimann et al. 1997). The normal outcome of antigen processing via this pathway is CTL tolerance, unless APC activation by CD4+ T-cells takes place (Kurts et al., 1997). To solve this problem of tolerance or functional deletion, WO 02/070006 disclosed the use of long HPV peptides as a vaccine, said peptide having both a MHC Class I and II presentable epitopes resulting in the activation of both CD4+ and CD8+ T-cells.
HPV vaccines developed in WO 02/070006 may still further be improved since high doses of peptides and/or sequential vaccinations are usually used in order to get an optimal immunogenic effect. Furthermore, adjuvants such as Montanide ISA-51 are usually required to get an optimal immunogenic effect. These adjuvants induce undesired side-effects such as prolonged local swelling at the site of injection, red swollen hands, fever, vomiting, joint pain, a general illness feeling similar to symptoms experienced during influenza infection. These side effects are generally experienced as uncomfortable and will prevent treatment of patients with early stage lesions.
Therefore, there is still a need for improved HPV vaccines, which do not have all the drawbacks of the existing vaccines, among other, the HPV vaccine used in the invention does not necessitate high doses of peptides and/or sequential vaccinations and/or an adjuvant.